Fragile X mental retardation protein (FMRP), encoded by the FMR1 gene on the X chromosome, is an RNA-binding protein that regulates the expression of hundreds of genes and plays a key role in brain development and function. The 5' untranslated region of the human FMR1 gene contains various numbers of CGG trinucleotide repeats. The modal number of repeats in the population is 30, whereas expansion of these CGG repeats above 200 leads to fragile X syndrome (FXS). CGG repeats between 55 and 200 are classified as ?premutation?, since these individuals are at increased risk for expansion into the FXS range. There is considerable polymorphism in the number of CGG repeats below 55, and controversy over the true definition of ?normal?. Indeed, the significance of either ?Gray Zone? (variously defined as 45-54 or 41-54) or ?Low Zone? (?23) CGG repeats, although much more prevalent than FXS and the premutation in the general population, are largely unexplored apart from the clinical literature. We have preliminary evidence to suggest that older adults with Low Zone repeats may be at increased risk of difficulties in cognitive and motor functioning as well as for having a child with a disability. We also found, in unpublished preliminary analyses, that Gray Zone expansions are associated with increased psychiatric and other health problems. Here we propose to validate and further explore these intriguing findings in a population-based cohort, a unique opportunity afforded by the Personalized Medicine Research Program (PMRP) of the Marshfield Clinic. In addition, we have established human embryonic stem cell (ESC) lines with defined numbers of CGG repeats. These lines, together with induced pluripotent stem cells (iPSC) derived from PMRP individuals with Low and Gray Zone CGGs, as well as controls, will provide an opportunity to assess whether having CGG repeats at either end of the distribution that currently is considered normal impairs expression, localization, and functions of FMRP. Building on our novel observations and unique collaborative opportunities, our two specific aims are to test the hypotheses that Low or Gray Zone CGG repeats in FMR1 are associated at the organismal level with compromised health and an increased risk of having a child with a disability; and at the cellular level with alterations in FMRP function. In Aim 1 we will determine, in a population-based sample, whether individuals with Low or Gray Zone CGG repeats are at increased risk of neurocognitive, motor, and physical and mental health problems, and having a child with a disability. We will use a combination of questionnaires and in-person assessments, as well as supervised machine learning algorithms on the comprehensive database of electronic health records available through the PMRP. In Aim 2 we will test the hypothesis that Low Zone or Gray Zone CGGs affect FMRP function and neural differentiation/maturation. We will use a combination of human ES cell lines engineered to have various CGG repeat lengths, and a series of induced pluripotent stem cell lines derived from patients with defined genotypes.